FALL 2014

Nice work Carolyn - great having you in the lab group this year! - DR. B 121214

WEEK 15

*Turned in final lab report, cleaned out personal box, and updated lab notebook.

WEEK 14

Analysis FPLC: The peak on the FPLC graph of Pv6PG was around 53kDa which is likely to be Pv6PG. The tubes collected were 31-34 to avoid obtaining anything that isn't Pv6PG. This sample was nanodropped to find the concentration and then spun down in a concentrating tube to 1ml and snap-freezed using liquid nitrogen to be stored in the -80 degree Celsius freezer.

Figure 3. Graphical representation of FPLC on Pv6PG with one small peak around 53 kDa,

12/03/14 Purification of Pv6PG

12042012- Fantastic work, keep it up!

WEEK 13

Figure 1. BL21(DE3) transformed competent cells with Pv6PG. Left: 50uL of BL21(DE3), 1uL Pv6PG plasmid and 200uL SOC media plated on LB agar + kanamycin plates after overnight incubation. Right: 50uL of BL21(DE3), 1uL Pv6PG plasmid and 50uL SOC media mixture plated on LB agar + kanamycin plates after overnight incubation. 11/18/14

WEEK 12

11/11/14 Making LB
4 liters of LB made for future large scale expressions. 2 expressions (each 2L) will be carried out side by side with one acting as a backup in case FPLC does not work out

For 2L LB
- 20g Bacto-tryptone
- 10g Bacto-yeast extract
- 20g NaCl

- Combine in 4L flask with 2000 mL of dH2O
- Stir until dissolve completely with stir bar
- Autoclaved

WEEK 11

FPLC

Figure 3. FPLC graph of Pv6PG showing two peaks in tubes 27-29 and 30-34. The peak furthest on the left is larger than 75kDa and the peak to the right of that is approximately 53kDa. 11/07/14

11/4/2014: Purification of Pv6PG. Samples 2-6 were stored in the 4 degree Celsius fridge.

Figure 2. Nanodrop of Elution 2 after Ni-NTA purification on the A280 Protein setting on the ND-1000 spectrophotometer. Results of Pv6PG Elution 2 samples after nickel column purification. The trial 1 concentration is 0.04mg/ml and the trial 2 concentration is 0.02 mg/ml. The average concentration is 0.03 mg/ml. The protein yield is measured to be 0.06mg.

Figure 1. Nanodrop of Elution 1 after Ni-NTA purification on the A280 Protein setting on the ND-1000 spectrophotometer. Results of Pv6PG Elution 1 samples after nickel column purification. The trial 1 concentration is 0.39mg/ml and the trial 2 concentration is 0.42 mg/ml. The average concentration is 0.405 mg/ml. The protein yield is measured to be 0.81mg.

WEEK 10

Figure 1. Sonication of Pv6PG after the pellet harvested by centrifugation and resuspended with 10ml of lysis buffer in a 15ml conical tube. 10/31/14.

Figure 1. Agarose gel of Nikki's PCR Cleanup from 9/22/14. Lane one contains 5mL of 1 kb ladder and lane 2 contains 10uL of sample with 2mL of blue juice. 10/27/14.

WEEK 7

Figure 5. PCR^2 on the secondary PCR samples from Figure 3. 1kb ladder in lanes 1and 6. Lanes 2-5 contain tubes A-D, respectively, for Sample 1 of secondary PCR. Lane 7-10 are the tubes A-D for Sample 2 of secondary PCR. 10/10/14

WEEK 6

Figure 4. PCR^2 on the secondary PCR samples from Figure 3. 1kb ladder in lanes 1and 6. Lanes 2-5 contain tubes A-D, respectively, for Sample 1 of secondary PCR. Lane 7-10 are the tubes A-D for Sample 2 of secondary PCR. 10/1/14

Analysis: Figures 4 and 5 show that the forward and reverse primers might not have been fully attached which left a lot of small pieces of DNA to run down the gel.

WEEK 5

Figure 3. Secondary PCR on the PCR cleanup sample of Pv6PG from summer. Lane 6 contains a 1kb ladder. Lane 7and contains Sample 1 and Lane 8 contains sample 2 both with 20uM forward and reverse primers for Pv6PG. 9/26/14

Analysis: The contamination shown in Figure 3 may have been caused by removing the comb out of the gel before it was completely dry. Lanes 1-5 were not usable.

Figure 2. Agarose gel for secondary PCR on the summer PCR cleanup sample for Pv6PG. Lanes 1 and 4 contain a 1kb ladder. Lanes 2 and 3 contain 12uL of sample both with the 20mM forward and reverse primers for Pv6PG. 9/26/14

Analysis: The gel was blank because instead of using EtBR, 0.5uL of red juice from a green PCR tube was used.

Figure 1. Agarose gel for Pv6PG with a 1kb ladder in lanes 1 and 3. Lane 2 contains 12uL of PCR cleanup sample from the sample. 9/22/14

09242014- Good job, but don't forget to give an analysis and leave captions

Figure 3. Agarose gel using secondary PCR for Pv6PG. Five different 1kb ladders (from the eppendorf tubes) in lanes 1-3 and 5-6. Lane 4 has 12uL of sample (10uL of secondary PCR and 2uL of gel loading dye). No bands appeared in Lane 4.

After two gels my secondary PCR has not worked. I think I need to go back and redo my oligo mix.

Figure 2. Secondary PCR of Pv6PG with a 5 uL of 1kb in both lanes 1 and 3. 18uL of sample in lane 2. No bands shown in any lane. 9/11/14

Figure 1. Primary PCR of Pv6PG with a 5uL of 1kb ladder in lane 1 and 18uL of sample in lane 2. 9/20/14.

Although it seems primary PCR worked in Figure 1 no bands for both the ladder and the sample appeared under the UV light. This means that my sample doesn't actually work, or my gel was not good because the ladder didn't show up as well.

Week 7

7/17/14 PCR Cleanup

Figure 1. DNA yields from PCR Cleanup after nanodropping. Trial 1: 50.1 ng/uL; Trial 2: 54.7 ng/uL

07/17/14 PCR Squared

Figure 2. PCR^2 Agarose Gel for Pv6PG. Lane 1: 1 kb ladder; Lane 2: Sample A #1; Lane 3: Sample A #2; Lane 4: Sample A #3; Lane 5: Sample A #4; Lane 6: Empty; Lane 7: Sample B #1; Lane 8: Sample B #2; Lane 9: Sample B #3; Lane 10: Sample B #4. Sample A was made at 55 degrees Celsius during Secondary PCR and Sample B was made at 55.8 degrees Celsius.

Figure 3. Temperature gradient for Secondary PCR. Samples were placed from the lower temperature to higher temperatures

^*Runs 24-31

Figure 6: Secondary PCR with the elongation time increased to 50 seconds and following the temperature gradient

Week 6

Keep up the good work! You'll get it soon (: Which primers were wrong? Include a virtual gel of RE Digest to [[#|verify]]. -Grace
7/15/14
___

Figure 1. PCR Agarose gel of pNIc-Bsa4 with no bands shown in Lanes 2-5, 7-10. Lane 1: 1 kb ladder; Lane 2: Sample A (Chris); Lane 3: Sample B (Chris); Lane 4: Sample C (Chris); Lane 5: Sample D (Chris); Lane 6: Empty; Lane 7: Sample A (Carolyn; 1 uL of 1:1000 stock dilution); Lane 8: Sample B (Carolyn; 1 uL of 1:100 stock dilution); Lane 9: Sample C (Carolyn; 1 uL of 1:10 stock dilution); Lane 10: Sample D (Carolyn; Control, no DNA template) 07/07/14

PCR for pNIC-Bsa4 has not worked after three times. A fourth time will be attempted later using different [[#|primers]].

Figure 2. PCR Agarose gel of pNIc-Bsa4 #4 with no bands shown in Lanes 2-5. Lane 1: 1 kb ladder; Lane 2: Sample A (Carolyn; 1 uL of 1:1000 stock dilution); Lane 3: Sample B (Carolyn; 1 uL of 1:100 stock dilution); Lane 4: Sample C (Carolyn; 1 uL of 1:10 stock dilution); Lane 5: Sample D (Carolyn; Control, no DNA template) 7/10/14

Figure 3. Agarose gel for primary PCR. Lane 1: 1 kb DNA ladder; Lane 2: Primary PCR of oligomix (Carolyn & Nikki); Lane 3: Primary PCR of oligomix (Zain & Luis); Lane 4: Primary PCR of oligomix (Hailey); Lane 5: Primary PCR of oligomix (Hailey-2); Lane 6: 100bp DNA ladder; Lane 7: 1 kb DNA ladder

*Smears are okay for primary PCR because the primers had not been added to the bands did not separate

Figure 4. Agarose Gel of secondary PCR. Lane 1 contains the DNA 1 kb ladder, lane 2 has 12 ul secondary PCR sample (Nikki & Carolyn) and lane 3 has 12 ul secondary PCR sample (Cidia). 7/10/14.

Figure 5. RE digest PCR for pGBR22. Lane 1: Empty; Lane 2: 5uL of 1 kb ladder; Lane 3: 12uL of uncut plasmid (pGBR22 300ng/uL); Lane 4: Sample 1 with 1uL of EcoRI; Lane 5: Sample 2 with 1uL of PvuII; Lane 6: Sample 3 with 0.5uL of EcoRI and 0.5uL of PvuII

1 kb ladder from NE Biolabs

Results for RE digest are good but there are slight bands in lanes 5 & 6 under the 125ng band possible due to contamination?

Week 5

pNIC-Bsa4 PCR

Figure 1. PCR agarose gel for pNIC-Bsa4. Lane 1: 1kb ladder; Lane 2: Sample A with 1 uL of 1:1000 stock dilution; Lane 3: Sample B with 1 uL of 1:100 stock dilution; Lane 4: Sample C with 1 uL of 1:10 stock dilution; Lane 5: Control, no DNA template. 7/1/14.

Figure 2. PCR agarose gel for pNIC-Bsa4. Lane 1: 1kb ladder; Lane 2: Sample A with 1 uL of 1:1000 stock dilution; Lane 3: Sample B with 1 uL of 1:100 stock dilution; Lane 4: Sample C with 1 uL of 1:10 stock dilution. Lane 5: Control, no DNA template. 7/3/14.

Oligo Mix

Done on July 2nd. 1 uL sample from each of the 36 wells were placed in a micro centrifuge tube with 64 uL of autoclaved nanopure water and stored in my box in the -20 degree Celsius fridge. This mix will be used in primary PCR for our target.

FPLC YopH BL21

Figure 3. Results from the FPLC machine for YopH BL21 with the standard curves for reference (dotted lines).

7/3/14 - We concentrated the 33 kDa and 44 kDa samples to 1 ml. 0.5 ml of each of the samples were placed in the -80 degree Celsius freezer and the other 0.5 ml is in the -20 degree Celsius fridge.

Week 4

Figure 1. PCR Agarose gel for pGBR22. Lane 1: 100bp marker (5 uL); Lane 2: Sample D (control, no DNA template); Lane 3: Sample B (3 ng DNA template); Lane 4: Sample C (30 ng DNA template); Lane 5: Sample A (0.3 ng DNA template). Contamination seen in lane 4. Samples A and D were switched.

Figure 2. Protein purification of YopH BL21 using a Ni-NTA column

Figure 3. Nanodrop spectrophotometry of a 2 ul sample of the Elution 1 collected after purification of YopH BL21. n=2 at a 280nm wavelength with an average volume of 0.805 and an average 260/280 score of 0.59. Nanodrop yields are pre-FPLC.

Figure 4. Nanodrop spectrophotometry of a 2 ul sample of the Elution 2 collected after purification of YopH BL21. n=2 at a 280nm wavelength with an average volume of 0.13 and an average 260/280 score of 0.83. Nanodrop yields are pre-FPLC.

Figure 5. Making of the SDS Page Gel consisting of a separating gel and a stacking gel. Bromophenol blue powder was used to show the wells. Finished product was stored in the 4 degree Celsius fridge.

Figure 6: SDS Page Gel of YopH BL21 after destaining the gel and placed on Whatman filter paper and covered in cellophane. Contamination in lanes 2-5Lane 1: Ladder (Colorstain Prestained Protein 10-230kDa)Lane 2: cell lysate before induction (Sample 0)Lane 3: cell lysate after inducation (Sample 1)Lane 4: soluble fraction (Sample 2)Lane 5: flow though (Sample 3)Lane 6: wash (Sample 4)Lane 7: Elution 1 (Sample 5)Lane 8: Elution 2 (Sample 6)

Week 3

Protein Expression

Pellet weight: 3.7g

Midi Prep Results
Nanodrop

Figure 1. 2 uL of pGBR22 sample measured on the Nanodrop spectrophotometer at a 260nm wavelength under the nucleic acid option.Concentration measured at 44.6 ng/uL. n=2

Figure 2. 2 uL of pGBR22 sample measured on the Nanodrop spectrophotometer at a 260nm wavelength under the nucleic acid option.Concentration measured at 44.1 ng/uL. n=2

Midi Prep DNA Sequencing

Figure 3. DNA sequence from the IMCB Core LIMS website for pGBR22 requesting M13 forward-40 (top) and M13 reverse-38 primers (bottom)

Week 1 and 2

Figure 1. DNA sequence for pGBR22 obtained from the DNA Core LIMS website using the M13 forward-40 primer. The original DNA sequence (top), the sequence after rerunning using a difficult template buffer (middle), and sequence after resubmitting without adding the M13 primer.

Figure 2. Alignment obtained from the BLASTn website using the DNA sequence from Figure 1

Figure 3. Petri dish with LB Agar, Kanamycin, SOC media, and DH5alpha incubated for approximately 24 hours in a 37 degrees Celsius fridge serving as the control

Figure 4. Petri dish with LB Agar, Kanamycin, SOC media, and DH5alpha cells that were transformed with either 1, 5, or 25 ng of pNIC incubated for approximately 24 hours in a 37 degrees Celsius fridge. No visible growth.

Figure 5. Petri dish with LB Agar, Kanamycin, SOC media, and DH5alpha cells that were transformed with either 1, 5, or 25 ng of pNIC incubated for approximately 24 hours in a 37 degrees Celsius fridge. No visible growth.

Figure 6. Petri dish with LB Agar, Kanamycin, SOC media, and DH5alpha cells that were transformed with either 1, 5, or 25 ng of pNIC incubated for approximately 24 hours in a 37 degrees Celsius fridge. No visible growth.

Carolyn T. (RP Summ 14)